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Excessive HIF-1α driven by phospholipid metabolism causes septic cardiomyopathy through cytopathic hypoxia

Nature Cardiovascular Research volume 4, pages 1077–1093 (2025)Cite this article

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Abstract

Septic cardiomyopathy, one manifestation of multiple organ dysfunction syndrome, is a challenging complication in sepsis, and cytopathic hypoxia has been proposed to have a key role in the pathophysiology of multiple organ dysfunction syndrome. However, the underlying mechanisms remain unknown. Here, we show that upregulation of hypoxia-inducible factor-1α (HIF-1α) in cardiomyocytes following lipopolysaccharide (LPS) treatment suppresses mitochondrial respiration via inducible nitric oxide synthase-dependent nitric oxide, leading to cytopathic hypoxia. Cardiac-specific heterozygous deletion of HIF-1α ameliorates mitochondrial and contractile dysfunction in a mouse model of septic cardiomyopathy. Mechanistically, nuclear factor-κB (NF-κB)-mediated upregulation of cyclooxygenase 2 (COX2) and secretory phospholipases A2 (sPLA2) enhances HIF-1α expression following LPS exposure, whereas their inhibition prevents LPS-induced HIF-1α upregulation, cytopathic hypoxia and contractile dysfunction. In addition, phospholipid metabolites (prostaglandins and lysophospholipids/free fatty acids, respectively) stabilize HIF-1α via protein kinase A activation. These findings highlight a crucial role of excessive HIF-1α, driven by LPS-enhanced phospholipid metabolism, in septic cardiomyopathy through induction of cytopathic hypoxia.

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Fig. 1: HIF-1α upregulation and cytopathic hypoxia in the myocardium of a septic cardiomyopathy model and cultured cardiomyocytes under LPS treatment.
Fig. 2: HIF-1α-induced cytopathic hypoxia and the role of nitric oxide.
Fig. 3: Effect of cardiac-specific HIF-1α heterozygous deletion on left ventricle contractility and complex II activity.
Fig. 4: Role of COX2 in LPS-induced HIF-1α upregulation and cytopathic hypoxia.
Fig. 5: Role of PGE2 in COX2-induced HIF-1α upregulation and septic cardiomyopathy.
Fig. 6: Role of sPLA2 in LPS-induced HIF-1α upregulation and cytopathic hypoxia.
Fig. 7: Role of sPLA2 in LPS-induced HIF-1α upregulation and septic cardiomyopathy.
Fig. 8: Role of protein kinase A in LPS-induced HIF-1α upregulation and septic cardiomyopathy.

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Data availability

The microarray data have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE297683. Owing to concerns regarding potential misuse or unauthorized secondary use, all other raw data will be made available upon reasonable request. Requests should be directed to the corresponding author, M.I.

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Acknowledgements

The authors thank M. Sato for her technical assistance. This work was supported by the Japan Society for the Promotion of Science KAKENHI (grant numbers JP24K22274 and JP24K02449 to M.I. and JP23H05481 to K.-I.Y.), the Japan Foundation for Applied Enzymology (Vascular Biology of Innovation to M.I.), MSD Life Science Foundation, the Public Interest Incorporated Foundation (to M.I.), SENSHIN Medical Research Foundation (to M.I.), The Cardiovascular Research Fund, Tokyo, Japan (to M.I.), and The Yomiuri Telecasting Charity Fund Research Grant (to M.I.).

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  1. Deceased: Tomomi Ide.

Authors and Affiliations

  1. Department of Cardiovascular Medicine, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan

    Masatsugu Watanabe, Masataka Ikeda, Ko Abe, Shun Furusawa, Kosei Ishimaru, Takuya Kanamura, Satoshi Fujita, Hiroko Deguchi Miyamoto, Yoko Shojima Isayama, Yuki Ikeda, Takashi Kai, Toru Hashimoto, Shouji Matsushima, Tomomi Ide, Hiroyuki Tsutsui & Kohtaro Abe

  2. Division of Cardiovascular Medicine, Research Institute of Angiocardiology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan

    Masatsugu Watanabe, Masataka Ikeda, Ko Abe, Shun Furusawa, Kosei Ishimaru, Takuya Kanamura, Satoshi Fujita, Hiroko Deguchi Miyamoto, Yoko Shojima Isayama, Yuki Ikeda, Takashi Kai, Toru Hashimoto, Shouji Matsushima, Tomomi Ide, Hiroyuki Tsutsui & Kohtaro Abe

  3. Department of Anesthesiology and Critical Care Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

    Masatsugu Watanabe & Ken Yamaura

  4. Department of Cardiovascular Medicine, Kyushu University Beppu Hospital, Oita, Japan

    Masataka Ikeda

  5. Department of Molecular Pathobiology, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan

    Eisho Kozakura & Ken-ichi Yamada

  6. School of Medicine and Graduate School, International University of Health and Welfare, Fukuoka, Japan

    Hiroyuki Tsutsui

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Contributions

M.W. was responsible for data curation, formal analysis, investigation, methodology, visualization, validation and writing the original draft. M.I. was responsible for conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, supervision, validation, visualization, writing the original draft and review and editing. Ko Abe, S. Furusawa, K.I., T. Kanamura., S. Fujita, H.D.M., E.K., Y.S.I., Y.I. and T. Kai were responsible for investigation. T.H., S.M., T.I. and H.T. were responsible for review and editing. K.-I. Y., K.Y. and Kohtaro Abe were responsible for supervision and review and editing.

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Correspondence to Masataka Ikeda.

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Extended data

Extended Data Fig. 1 A murine model of septic cardiomyopathy.

(a) Left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) of C57BL/6 J mice 6 h post-LPS administration (n = 3, each group). (b) Echocardiographic images of the left ventricle of C57BL/6 J mice 24 h post-LPS administration. (c) Left ventricular ejection fraction (LVEF), LVESD, and LVEDD of C57BL/6 J mice 24 h post-LPS administration (n = 3 in the control [Ctrl] group and n = 4 in the LPS group). (d) Hif1a gene expression in the myocardium of C57BL/6 J mice 24 h post-LPS administration, treated with LPS (n = 3 in the Ctrl group and n = 5 in the 24 h group). (e) Western blot analysis of HIF-1α expression in the myocardium of C57BL/6 J mice 24 h post-LPS administration and its quantification (n = 6, each group). GAPDH was used as an internal control. Data are presented as the mean ± SEM and analyzed using a two-sided t-test. *P < 0.05, **P < 0.01.

Source data

Extended Data Fig. 2 Biological and histological features of the septic cardiomyopathy model.

(a) Blood pH of mice 6 h after LPS administration (n = 3 in the control [Ctrl] group and n = 4 in the LPS group). (b) Malondialdehyde (MDA) 6 and 24 h after LPS administration (n = 3 in the Ctrl group, n = 3 in the LPS 6 h group, and n = 5 in the LPS 24 h group). (c) Western blot analysis of acrolein-modified proteins in mice 6 and 24 h after LPS administration and their quantifications (n = 3 in the Ctrl group, n = 3 in the LPS 6 h group, and n = 5 in the LPS 24 h group). (d) Hematoxylin/eosin staining of the myocardium 6 and 24 h after LPS administration (n = 6 in Ctrl group, n = 6 in the LPS 6 h group, and n = 3 in the LPS 24 h group). Scale bars, 50 μm. (e) Western blots of CD3, B220, and CD107b in mice 6 and 24 h after LPS administration and their quantifications (n = 3 in the Ctrl group, n = 3 in the LPS 6 h group, and n = 5 in the LPS 24 h group). (f–h) Gene expression of inflammatory cytokines, such as Il1b, Il6, and Tnf, in the myocardium 6 and 24 h after LPS administration (n = 3 in the Ctrl group, n = 3 in the LPS 6 h group, and n = 5 in the LPS 24 h group). GAPDH was used as an internal control. Data are presented as the mean ± SEM. Data were statistically analyzed using a two-sided t-test (a) and two-sided Dunnett’s test (b–h). *P < 0.05, **P < 0.01.

Source data

Extended Data Fig. 3 HIF-1α upregulation and impaired mitochondrial respiration in a LPS dose-dependent manner.

(a) Hif1a gene expression in cultured cardiomyocytes treated with LPS (1, 10, 100 ng/mL; n = 6, each group). (b) Western blot analysis of HIF-1α expression in cultured cardiomyocytes treated with LPS (10, 100, 1000 ng/mL) and their quantifications (n = 6, each group). GAPDH was used as an internal control in western blotting experiments. (c) Overall flow of the Mitostress test using the Flux Analyzer in cultured cardiomyocytes treated with LPS (10, 100, 1000 ng/mL; n = 4, each group). (d) Summary of oxygen consumption rates (OCRs) in the Mitostress test shown in panel (c) (n = 4, each group). Mitochondrial reserve capacity (MRC) was calculated by subtracting basal OCR from maximal respiration OCR shown in panel (c). (e) Hif1a gene expression in isolated adult mouse cardiomyocytes treated with LPS (100 ng/mL, 24 h) (n = 4, each group). (f) Maximal OCR under FCCP treatment and Rotenone (Rot)/antimycin (AMA) using the Flux Analyzer in isolated adult mouse cardiomyocytes treated with LPS (n = 4, each group). Data are presented as the mean ± SEM and analyzed using a two-sided Dunnett’s test (a and b), one-way ANOVA with Tukey’s post hoc test (d), and two-sided t-test (e and f). *P < 0.05, **P < 0.01.

Source data

Extended Data Fig. 4 Roles of TLR4, inflammatory cytokines, and NO in HIF-1α upregulation and cytopathic hypoxia in cardiomyocytes under LPS treatment.

(a and b) Western blot analysis of HIF-1α expression in cultured cardiomyocytes treated with LPS (a, 100 ng/mL; n = 6, each group) or lipoteichoic acid (LTA) (b, 50 μg/mL; n = 3, each group) and TAK-242 (1 μM). (c) Overall flow of the Mitostress test using the Flux Analyzer (upper) and summary of oxygen consumption rates (OCRs) in the Mitostress test (lower) in cultured cardiomyocytes treated with LPS (100 ng/mL; n = 4, each group) and TAK-242 (1 μM). (d) Overall flow of the Mitostress test using the Flux Analyzer (upper) and summary of OCRs in the Mitostress test (lower) in cultured cardiomyocytes treated with LTA (50 μg/mL; n = 4, each group) and TAK-242 (1 μM). Mitochondrial reserve capacity (MRC) was calculated by subtracting basal OCR from maximal respiration OCR. (e) Western blot analysis of HIF-1α in cultured cardiomyocytes treated with IL-1β (10 ng/mL, 24 h) (left), IL-6 (10 ng/mL, 24 h) (middle), and TNF (3 ng/mL, 24 h) (right) (n = 6, each group). (f and g) Overall flow of the Mitostress test using the Flux Analyzer in LPS-treated cultured cardiomyocytes, in which inducible NOS (iNOS) was silenced by specific siRNA (f; n = 4, each group) or NO was scavenged by 100 μM C-PTIO (g; n = 8, each group). GAPDH was used as an internal control in western blotting experiments. Data are presented as the mean ± SEM. Data were statistically analyzed using 1-way ANOVA with Tukey’s post hoc test (a–d) and a two-sided t-test (e). *P < 0.05, **P < 0.01.

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Extended Data Fig. 5 Echocardiographic data and the myocardial expression of HIF-1α in control [Ctrl] mice and cardiomyocyte-specific HIF-1α hetero knockout (caHetKO) mice 24 h after LPS treatment.

(a) Echocardiographic images of the left ventricle of Ctrl and caHetKO mice 24 h after LPS administration. (b) Left ventricular ejection fraction (LVEF, left), left ventricular end-systolic diameter (LVESD, middle), and left ventricular end-diastolic diameter (LVEDD, right) in Ctrl and caHetKO mice 24 h after LPS administration (n = 8, each group). (c) Western blot analysis of HIF-1α expression in the myocardium of Ctrl and caHetKO mice 24 h after LPS administration and its quantification (n = 8, each group). GAPDH was used as an internal control. Data are presented as the mean ± SEM and analyzed using a two-sided t-test. **P < 0.01.

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Extended Data Fig. 6 Roles of the NF-κB family in cultured cardiomyocytes under LPS treatment.

(a) Western blots of p105/p50 and HIF-1α expression under LPS (100 ng/mL) treatment in cultured cardiomyocytes with or without Nfkb1 silencing and quantification of HIF-1α (n = 6, each group). (b) Hif1a gene expression in cultured cardiomyocytes treated with siRNA against Nfkb1 (n = 9, each group). (c) Western blots of RELA and HIF-1α expression under LPS treatment in cultured cardiomyocytes with or without Rela silencing and quantification of HIF-1α (n = 6, each group). (d) Hif1a gene expression in cultured cardiomyocytes treated with siRNA against Rela (n = 6, each group). (e) Western blots of c-Rel and HIF-1α expression under LPS treatment in cultured cardiomyocytes with or without Rel silencing and quantification of HIF-1α (n = 6, each group). (f) Hif1a gene expression in cultured cardiomyocytes treated with siRNA against Rel (n = 9, each group). (g) Western blots of p100/p52 and HIF-1α expression under LPS treatment in cultured cardiomyocytes with or without Nfkb2 silencing and quantification of HIF-1α (n = 3, each group). (h) Western blots of RelB and HIF-1α expression under LPS treatment in cultured cardiomyocytes with or without Relb silencing and quantification of HIF-1α (n = 3, each group). GAPDH was used as an internal control in western blotting experiments. Data are presented as the mean ± SEM and analyzed using 1-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01.

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Extended Data Fig. 7 Roles of COX2 and secretory phospholipase A2 (sPLA2) in LPS-induced HIF-1α expression.

(a–b) Overall flow of the Mitostress test using the Flux Analyzer in LPS (100 ng/mL)-treated cultured cardiomyocytes, in which COX2 was silenced by specific siRNA (a) (n = 4, each group) or inhibited by flurbiprofen (b) (n = 8, each group). (c) Hif1a gene expression in cultured cardiomyocytes treated with prostaglandin E2 (10 μM, 24 h) (left; n = 4) and prostaglandin I2 (100 μM, 24 h) (right; n = 4). (d) Western blot analysis of HIF-1α in cardiomyocytes treated with U-46619 (30 μM, 24 h), an agonist for the thromboxane A2 receptor, and its quantification (n = 6, each group). GAPDH was used as an internal control in western blotting experiments. Data are presented as the mean ± SEM and analyzed using a two- sided t-test.

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Extended Data Fig. 8 Roles of secretory phospholipase A2 (sPLA2) and lysophosphatidylcholine (lysoPC) in septic cardiomyopathy.

(a) Ptgs2 gene expression in cultured cardiomyocytes treated with LPS (100 ng/mL) and siRNA targeting Nfkb1, Rela, and Rel (n = 6 per group). (b) Enpp2 gene expression in cultured cardiomyocytes treated with LPS and siRNA against Nfkb1, Rela, and Rel (n = 4 in the siNfkb1 group, n = 4 in the siRela group, and n = 3 in the siRel group). (c and d) Western blots of autotaxin (ATX) and HIF-1α expression under LPS treatment in cultured cardiomyocytes with Enpp2 silencing and their quantifications (n = 3, each group). GAPDH was used as an internal control in western blotting experiments. (e) Overall flow of the Mitostress test using the Flux Analyzer in LPS-treated cultured cardiomyocytes, in which PLA2G2A was silenced by specific siRNA (n = 4, each group). (f) Experimental protocol for treatment of C57BL/6 J mice with Varespladib (an inhibitor against PLA2G2A, PLA2G5, and PLA2G10) under LPS administration. (g) Echocardiographic images of the left ventricles of C57BL/6 J mice with VPL treatment 6 h after LPS administration. (h) LVEF of C57BL/6 J mice 6 h after LPS administration with or without VPL treatment (n = 6, each group). (i) Western blot analysis of HIF-1α expression in the myocardium of C57BL/6 J mice 6 h after LPS administration with or without VPL and its quantification (n = 6, each group). GAPDH was used as an internal control in western blotting experiments. (j) The levels of lysoPCs (left, 16:0; middle, 18:0; right, 18:1) in the blood of mice treated with PLA2G5-neutralizing antibody, 6 h after LPS administration (n = 6, each group). (k) Western blot of autotaxin (ATX) expression in the plasma of C57BL/6 J mice 6 h after LPS administration and its quantification (n = 6, each group). Coomassie Brilliant Blue (CBB) was used as an internal control in western blotting experiments. Data are presented as the mean ± SEM. Data were statistically analyzed using a two-sided t test (a, c, k) and 1-way ANOVA with Tukey’s post hoc test (d, h–j). *P < 0.05, **P < 0.01.

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Extended Data Fig. 9 Roles of protein kinase A (PKA) in HIF-1α upregulation in cardiomyocytes, which is induced by prostaglandin E2 (PGE2), lysophosphatidylcholine (lysoPC), and oleic acid (OA) and HIF-2α in cultured cardiomyocytes treated with LPS.

(a) Western blot analysis of HIF-2α in cultured cardiomyocytes, in which HIF-2α was silenced by specific siRNA (n = 3, each group). (b) Overall flow of the Mitostress test using the Flux Analyzer in LPS-treated cultured cardiomyocytes, in which HIF-2α was silenced by specific siRNA. (c) Summary of oxygen consumption rates (OCRs) in the Mitostress test shown in panel (b) (n = 4, each group). Mitochondrial reserve capacity (MRC) was calculated by subtracting basal OCR from maximal respiration OCR shown in panel (e). (d–f) Western blot analysis of HIF-1α in cultured cardiomyocytes treated with PGE2 (10 μM, 24 h) (d), lysoPC (200 μM [100 μg/mL], 3 h) (e), and OA (30 μM, 24 h) (f) (n = 6, each group). H-89 (30 μM) was co-treated for an inhibition of PKA. GAPDH was used as an internal control in western blotting experiments. Data are presented as the mean ± SEM and analyzed using one-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01.

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Extended Data Fig. 10 Graphical abstract depicting the present findings.

COX2, cyclooxygenase 2; FFA, free fatty acid; iNOS, inducible nitric oxide synthase; NO, nitric oxide; LPS, lipopolysaccharide; LTA, lipoteichoic acid; PG, prostaglandin; PKA, protein kinase A; TLR4, toll-like receptor-4.

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Watanabe, M., Ikeda, M., Abe, K. et al. Excessive HIF-1α driven by phospholipid metabolism causes septic cardiomyopathy through cytopathic hypoxia. Nat Cardiovasc Res 4, 1077–1093 (2025). https://doi.org/10.1038/s44161-025-00687-1

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